Navigation Links
Pathway toward gene silencing described in plants

Biologists at Washington University in St. Louis have made an important breakthrough in understanding a pathway plant cells take to silence unwanted or extra genes using short bits of RNA. Basically, they have made it possible to see where, and how, the events in the pathway unfold within the cell, and seeing is believing, as the old saying goes.

Craig Pikaard, Ph.D., Washington University professor of biology in Arts & Sciences and his collaborators have described the roles that eight proteins in Arabidopsis plants play in a pathway that brings about DNA methylation, an epigenetic function that involves a chemical modification of cytosine, one of the four chemical subunits of DNA. Without proper DNA methylation, higher organisms from plants to humans have a host of developmental problems, from dwarfing in plants to certain tumors in humans, and death in mice. One role of DNA methylation is to turn off repetitive genes, such as transposable elements that can move or spread throughout a genome and disrupt other gene functions if left unchecked. There is also interest in DNA methylation because understanding how some genes are selectively silenced and how silenced alleles can be turned on again may someday have practical benefits. For instance, tumor suppressor genes that normally help keep cells from dividing uncontrollably are often silenced by DNA methylation and histone (proteins that wrap DNA) modifications in cancer cells, contributing to tumor growth. And certain blood disorders resulting from defective genes expressed in adults might be alleviated if versions of those same genes that are only expressed very early in development, but are then silenced in adults, could only be turned on again.

"The pathway we are studying is part of an interesting phenomenon that occurs in plants, and reportedly in humans, too, called RNA-directed DNA methylation," Pikaard explained. "This pathway takes place in the nucleus, and it involves short RNA s, called small interfering RNAs -- siRNAs."

Those little tykes, just 24 nucleotides long, are somehow responsible for methylation of DNA sequences that match the sequence of the siRNAs, but not without a lot of help from their friends. The friends in this case are the team of eight known proteins of the RNA-directed DNA methylation pathway.

Using an impressive toolkit of sophisticated techniques, Pikaard and his collaborators not only have described the locations of the eight proteins in the pathway but also have provided the sequence of events that leads to methylation. It is a twisted, and ultimately circular path, but Pikaard and his colleagues are the first researchers to literally see the pathway and thereby provide a clearer understanding of the steps leading to methylation and gene silencing.

The results were published in the July 14, 2006 issue of Cell. The study was funded by the National Institutes of Health, Howard Hughes Medical Institute (HHMI) and Monsanto Company. Pikaard's collaborators include Olga Pontes, the first author of the study, other group members from his Washington University laboratory and the group of Steven E. Jacobsen, Ph.D., an HHMI investigator and professor of biology at the University of California, Los Angeles.

Using mutants, antibodies, and fluorescence microscopy techniques known as RNA fluorescence in situ hybridization (RNA-FISH) and DNA-FISH, Washington University postdoctoral researcher Olga Pontes, Ph.D., was able to unravel where the eight team players are located and in what order events in the RNA-directed DNA methylation pathway transpire. Using antibodies to detect the proteins, together with DNA-FISH to detect the DNA sites that give rise to the siRNAs, Pontes found that half of the team is located with the genes that match the siRNAs.

"The combination of DNA FISH and protein localization allowed us to say which proteins are sitting on the DNA that give rise to th e siRNAs and also the loci that are modified by the siRNAs," Pikaard said.

Pontes found the other half of the team located within a special nuclear compartment known as the nucleolus, long known to be the production center for ribosomes. "She got a brilliant signal in the nucleolus, a brilliant dot in the same place for each of the proteins," said Pikaard. Using RNA-FISH, Pontes also found that the siRNAs were in that same dot within the nucleolus.

Pontes and Pikaard were able to deduce the order of events by studying mutations of all eight genes that give rise to the proteins, finding out what happens to the different proteins as the different genes are mutated, one by one. For instance, the researchers found the importance of RNA Polymerase IVa (Pol IVa) by looking at a Pol IVa mutant and noting that the rest of the proteins didn't localize properly. In the RNA-dependent RNA polymerase 2 (RDR2) mutant, Pol IVa is unaffected, but the function of all the other proteins downstream is lost, inferring that it came into the act second. The picture that emerged from this logical approach is that Pol IVa gets things started, churning out RNA that then goes to the nucleolus where it is acted on by RDR2, which turns the single-stranded RNA into double-stranded RNA. The Dicer-like 3 protein, DCL3 then chops the RNA into small interfering RNAs (siRNAs). Along comes ARGONAUTE4 (AGO4), which grabs hold of the siRNAs while also binding to NRPD1b, the largest subunit of an alternative form of RNA Polymerase IV, Pol IVb. The AGO4-siRNA-NRPD1b complex is then thought to leave the nucleolus, acquire the second-largest Pol IV subunit, NRPD2, which serves both Pol IVa and Pol IVb, and then seek out the DNA sequences that match the siRNAs. At these sites, the chromatin remodeler DRD1 presumably bulldozes histones and other proteins out of the way to make the DNA accessible for methylation by the de novo cytosine methyltransferase, DRM2.

A paradoxical aspect of the pathway is that siRNAs direct DNA methylation but DNA methylation is also required for the production siRNAs. "It's a circular pathway. You have to produce the siRNA in order to have them come back and methylate the loci, which somehow induces more siRNA production involving Pol IVa". Pikaard said.

A combination of genetic mutants, transgenes, antibodies, RNA-FISH and DNA-FISH were key to the study. "This toolkit is really powerful," Pikaard said.

"It enabled us to look at a complex pathway and figure out not only the order of events but also the spatial organization of the pathway in the nucleus. Our hope for the future is to develop tools that will enable us to watch the pathway function in live cells using fluorescent proteins and time-lapsed microscopy, to learn even more."


Source:Washington University in St. Louis

Related biology news :

1. Breast Cancer May Be Uniquely Sensitive To Inhibitors Of PI3K Pathway
2. Measuring Enzymes At End Of Cancer Pathway Predicts Outcome Of Tarceva, Taxol
3. Ariadne Genomics Announces the Release of PathwayStudio?Central, Client-Server Software for Biological Pathway Analysis
4. Unchecked DNA replication drives earliest steps toward cancer
5. Virologists make major step towards understanding the process of HIV infection
6. Moffitt-USF head toward first human trials of anti-cancer drug that targets protein AKT
7. Cats indifference towards sugar explained
8. A step toward the $1,000 personal genome using readily available lab equipment
9. A new step towards an AIDS vaccine
10. New GM mosquito sexing technique is step towards malaria control, report scientists
11. Novel method reveals how menthol discovery could point towards new or improved pain therapies
Post Your Comments:

(Date:10/26/2015)... PALO ALTO, Calif. and LAS ... – Nok Nok Labs , an innovator in ... FIDO Alliance , today announced the launch of its ... the first unified platform enabling organizations to use standards-based ... authentication. The Nok Nok S3 Authentication Suite is ideal ...
(Date:10/23/2015)... Oct. 23, 2015 Research and Markets ( ... "Global Voice Recognition Biometrics Market 2015-2019" report to ... --> The global voice recognition biometrics market to ... --> --> The ... prepared based on an in-depth market analysis with inputs ...
(Date:10/22/2015)...  Aware, Inc. (NASDAQ: AWRE ), a leading supplier of ... quarter ended September 30, 2015.  --> ... $4.0 million, a decrease of 33% compared to $6.0 million in ... of 2015 was $2.2 million, or $0.10 per diluted share, which ... same period a year ago.  --> ...
Breaking Biology News(10 mins):
(Date:11/24/2015)... Nov. 24, 2015 /PRNewswire/ - Aeterna Zentaris Inc. ... that the remaining 11,000 post-share consolidation (or 1,100,000 ... (the "Series B Warrants") subject to the previously ... November 23, 2015, which will result in the ... effect to the issuance of such shares, there ...
(Date:11/24/2015)... ... November 24, 2015 , ... In harsh ... Insertion points for in-line sensors can represent a weak spot where leaking process ... series of retractable sensor housings , which are designed to tolerate extreme process ...
(Date:11/24/2015)... ... November 24, 2015 , ... Creation Technologies would like to ... Deloitte's 2015 Technology Fast 500 list of the fastest growing companies in North ... II medical device that speeds up orthodontic tooth movement by as much as ...
(Date:11/24/2015)... Capricor Therapeutics, Inc. (NASDAQ: ... development and commercialization of first-in-class therapeutics, today announced that ... scheduled to present at the 2015 Piper Jaffray Healthcare ... at The Lotte New York Palace Hotel in ... . --> . ...
Breaking Biology Technology: